EP4034673A1 - Procédés, compositions et kits pour déterminer le sexe d'un foetus - Google Patents

Procédés, compositions et kits pour déterminer le sexe d'un foetus

Info

Publication number
EP4034673A1
EP4034673A1 EP20867331.9A EP20867331A EP4034673A1 EP 4034673 A1 EP4034673 A1 EP 4034673A1 EP 20867331 A EP20867331 A EP 20867331A EP 4034673 A1 EP4034673 A1 EP 4034673A1
Authority
EP
European Patent Office
Prior art keywords
days
sample
blood
weeks
nucleic acids
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20867331.9A
Other languages
German (de)
English (en)
Other versions
EP4034673A4 (fr
Inventor
Christopher Jacob
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Gateway Genomics LLC
Original Assignee
Gateway Genomics LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gateway Genomics LLC filed Critical Gateway Genomics LLC
Publication of EP4034673A1 publication Critical patent/EP4034673A1/fr
Publication of EP4034673A4 publication Critical patent/EP4034673A4/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the disclosure relates to methods, compositions, and kits for the early determination of the sex of a fetus.
  • the disclosure also provides methods, compositions, and kits for detecting fetal nucleic acids in biological samples (e.g., cell-free fetal DNA).
  • Ultrasound imaging has been used safely for decades and is considered highly accurate for determining fetal sex at 18 to 20 weeks of gestation.
  • Amniocentesis may be used for determining fetal sex with high accuracy between 15 to 18 weeks gestational age but carries a miscarriage risk and is not available to most women.
  • NIPT non-invasive prenatal testing
  • NIPT non-invasive prenatal testing
  • NIPT All methods of NIPT require a minimum fetal fraction for accurate trisomy screening, commonly estimated at 4% (G. Ashoor et al. (2013) Ultrasound in Obstetrics & Gynecology 41:26-32). Since fetal fraction is generally less than 4% at earlier time points in pregnancy, NIPT is not generally used for prenatal testing prior to 11 weeks.
  • Fetal sex is determined at the time of conception, approximately 2 weeks gestational age. There is a need in the art for methods and kits useful for determining fetal sex between 2- and 11 -weeks gestational age. Additionally, there is a need in the art for compositions for detecting fetal nucleic acids in biological samples. The present disclosure meets some or all of these needs by providing highly accurate, noninvasive methods for determining fetal sex at earlier timepoints than any conventional method. The present disclosure further provides novel methods, assays, kits, and compositions for detecting fetal nucleic acids and determining fetal sex in early pregnancy (e.g., 4 to 8 weeks gestational age).
  • the present disclosure provides methods of determining the sex of a fetus in a pregnant subject, typically a human subject, comprising: obtaining a biological sample from the subject; and detecting fetal Y-chromosome nucleic acids in the sample, thereby determining the sex of the fetus.
  • the methods of the disclosure further comprise enriching the sample for fetal nucleic acids.
  • the enrichment is achieved by separating plasma from whole blood, by selectively capturing fetal nucleic acids from the biological sample, or by selectively degrading maternal nucleic acids in the biological sample.
  • the Y-chromosome nucleic acids are cell-free fetal nucleic acids (e.g., cffDNA). In some embodiments, the Y-chromosome nucleic acids are genomic fetal nucleic acids from a fetal cell (e.g., gfDNA).
  • the disclosure provides methods for isolating and concentrating fetal nucleic acids in biological samples from pregnant subjects, (e.g. human subjects).
  • the methods further comprise isolating fetal nucleic acids with a silica column or magnetic beads.
  • the methods further comprise concentrating the fetal nucleic acids in the biological sample using heat.
  • the methods further comprise isolating fetal nucleic acids by incubating the sample with an enzyme (e.g., proteinase K).
  • the methods, compositions, and kits of the disclosure provide optimal sensitivity, specificity, and accuracy for fetal sex determination.
  • the methods of the disclosure determine the sex of the fetus with at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% specificity.
  • the methods of the disclosure determine the sex of the fetus with at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sensitivity.
  • the methods of the disclosure determine the sex of the fetus with at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% accuracy.
  • the false positive rate of the methods and kits of the disclosure is less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 20%, or less than 25%.
  • the performance of the methods, compositions, and kits of the disclosure have been determined in multiple populations. In some embodiments, the performance of the methods, compositions, and kits of the disclosure have been determined in a population of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, and/or 1,000 or more pregnant subjects.
  • the methods, compositions, and kits of the disclosure may be used at various gestations ages of pregnancy.
  • the gestational age of the fetus is 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, lOweeks, 11 weeks, or 12 weeks.
  • the gestational age of the fetus is 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days 42 days, 49 days, 56 days, 63 days, 70 days, 77 days, or 84 days.
  • the disclosure provides methods, compositions, and kits for detecting Y- chromosome nucleic acids in a biological sample from a pregnant subject.
  • the detecting comprises performing microarray analysis, polymerase chain reaction (PCR), or sequencing.
  • the PCR is quantitative PCR (qPCR) or digital PCR.
  • the qPCR is a TaqMan assay.
  • the PCR is a nested PCR, a duplex PCR, or a multiplex PCR.
  • the reaction volume of the PCR is 2ul, 3ul, 4ul, 5ul, lOul, 15ul, 20ul, 25ul, or 50ul.
  • the mastermix used in the PCR contains Uracil N Glycosylase (UNG).
  • the methods further comprise interpreting data generated when detecting the Y-chromosome DNA.
  • the interpreting is performed using a machine learning algorithm, a cycle-threshold (CT) algorithm, or artificial intelligence.
  • CT cycle-threshold
  • the biological sample is incubated with a preservative.
  • the preservative is an anti-coagulant (e.g., ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), heparin), an antimicrobial (e.g., imidazolidinyl urea), a sugar, and/or an amino acid.
  • the preservative is a solid, a liquid, and/or a gel.
  • the sample is blood, plasma, serum, saliva, urine, and/or cervical mucus.
  • the sample is maternal blood, maternal plasma, or maternal serum.
  • the volume of the sample obtained from the subject is lOul to 10ml.
  • the volume of the sample used to detect Y-chromosome DNA is a microvolume.
  • the microvolume is about l,000ul, about 900ul, about 800ul, about 700 ul, about 600ul, about 500ul, about 400ul, about 300ul, about 200ul, about 150ul, about lOOul, about 50ul, about 25ul, about lOul.
  • the biological sample can be processed at any time after being collected from the subject. In some embodiments, the biological sample is processed within 1 hour, within 24 hours, or within 48 hours. In other embodiments, the biological sample is not processed for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 1 week, at least, 2 weeks, or at least 4 weeks.
  • At least 12 hours, at least 18 hours, at least 24 hours, or at least 36 hours elapses between a time of blood collection and a time of any testing or screening of a collected blood sample, and or isolation of the cell-free nucleic acids.
  • at least 24 hours elapses between a time of blood collection and a time of any testing or screening of a collected blood sample, and or isolation of the cell-free nucleic acids.
  • more than 24 hours elapses between a time of blood collection and a time of any testing or screening of a collected blood sample, and or isolation of the cell-free nucleic acids.
  • the methods and kits of the disclosure may include instructions for decontaminating the site on the pregnant subject where the sample will be collected.
  • the decontamination is performed by applying bleach to the site of collection, by applying an alcohol wipe to the site of collection, by treating the site of collection with ultra-violet light, by applying chlorhexidine gluconate, hydrogen peroxide, and/or iodine to the site of collection, by applying a brush (e.g., a nail brush) to the site of the collection.
  • a brush e.g., a nail brush
  • kits for obtaining a biological sample from a pregnant subject may comprise a blood collection tube, a lancet or a device useful for obtaining venous or capillary blood from the subject, a tourniquet, a bandage, an alcohol swab, a nail or skin brush, and instructions for using the kits.
  • the kits further comprise a decontaminating agent.
  • the decontaminating agent is bleach, an alcohol wipe, chlorhexidine gluconate, hydrogen peroxide, and/or iodine.
  • the device for obtaining venous or capillary blood is a lancet (e.g., BD Microtainer contact-activated lancet), a syringe, and/or a push-button blood collection device (e.g., a TAP device or a Tasso-SST device).
  • the biological sample is collected into a tube, onto a card, and/or a swab.
  • the present disclosure provides methods for detecting Y-chromosome DNA in biological samples from pregnant subjects.
  • a set of nucleic acid primers and/or probe are used to amplify and/or detect the Y-chromosome DNA in the sample.
  • Primers and probes used in the methods of the disclosure may target one or more targets or target regions on the Y- chromosome (e.g., a gene on the Y-chromosome).
  • the target on the Y- chromosome is SRY, DYS, or DAZ.
  • the methods use one or more targets on the Y-chromosome to detect Y-chromosome DNA in the sample.
  • the target is a DNA sequence that is present in one or more locations on the Y-chromosome.
  • Kits of the disclosure include instructions for collecting the sample at various gestational ages.
  • the instructions provide for sample collection at gestational age of 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks.
  • the gestational age of the fetus is 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days 42 days, 49 days, 56 days, 63 days, 70 days, 77 days, or 84 days.
  • the methods, compositions, and kits of the disclosure can be used to detect very small amounts of Y-chromosome DNA in a biological sample from a pregnant subject.
  • the methods of the disclosure can detect about 1 to 0.1 genomic equivalent of cffDNA in a sample, about 0.9 genomic equivalent of cffDNA in a sample, about 0.8 genomic equivalent of cffDNA in a sample, about 0.7 genomic equivalent of cffDNA in a sample, about 0.6 genomic equivalent of cffDNA in a sample, about 0.5 genomic equivalent of cffDNA in a sample, about 0.4 genomic equivalent of cffDNA in a sample, about 0.3 genomic equivalent of cffDNA in a sample, about 0.2 genomic equivalent of cffDNA in a sample, about 0.1 genomic equivalent of cffDNA in a sample.
  • the fetal fraction in the biological sample is about 4%, about 3%, about 2%, about 1% or less than 1%.
  • the disclosure provides a method comprising: a) obtaining or having obtained a maternal capillary blood sample comprising cell-free nucleic acids, wherein the volume of blood is 500 microliters or less, b) incubating the blood sample with a cell-free nucleic acid preservative, c) storing the blood sample in a tube for at least 1 day at ambient temperature prior to testing the blood sample, d) collecting 50 to about 250 microliters of plasma from the blood sample, e) isolating cell-free nucleic acids from the blood sample, f) detecting Y-chromosome DNA in the cell- free nucleic acids by performing quantitative PCR with all or a portion of the isolated cell-free nucleic acids, g) determining the sex of a fetus as male based on the detection of Y-chromosome DNA in the cell-free nucleic acids, wherein the gestational age of the fetus is 6 weeks to 8 weeks, wherein the accuracy
  • the disclosure provides a method comprising: a) obtaining or having obtained a maternal venous blood sample comprising cell-free nucleic acids, wherein the volume of blood is 10 milliliters or less, b) incubating the blood sample with a cell-free nucleic acid preservative, c) storing the blood sample in a tube for at least 1 day at ambient temperature prior to testing the blood sample, d) collecting about 50 microliters to about 5 milliliters of plasma from the blood sample, e) isolating cell-free nucleic acids from the blood sample, f) detecting Y-chromosome DNA in the cell-free nucleic acids by performing quantitative PCR with all or a portion of the isolated cell-free nucleic acids, g) determining the sex of a fetus is male based on the detection of Y- chromosome DNA in the cell-free nucleic acids, wherein the gestational age of the fetus is 6 weeks to
  • An aspect of the present disclosure is a method of determining the sex of a fetus in a pregnant subject, comprising: obtaining a biological sample from the subject; and detecting fetal Y- chromosome nucleic acids in the sample, thereby determining the sex of the fetus.
  • the method further comprises enriching the sample for fetal nucleic acids.
  • the enrichment is achieved by separating plasma from whole blood, by selectively capturing fetal nucleic acids from the biological sample, and/or by selectively degrading maternal nucleic acids in the biological sample.
  • the Y-chromosome nucleic acids are cell- free fetal nucleic acids or genomic fetal nucleic acids from a fetal cell.
  • the method further comprises isolating and concentrating the fetal nucleic acids.
  • the isolating is achieved with a silica column or magnetic beads.
  • the sex of the fetus is determined with at least 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% accuracy.
  • the performance of the method has been determined in a population of at least 50 or more pregnant subjects.
  • the gestational age of the fetus is selected from the group consisting of between 4 weeks and 8 weeks, not more than 56, 53, 49, 46, 42, 39, 35, 32, or 28 days or a range defined by any two of the preceding values, 35-53 days, 42-49 days and 42-53 days.
  • the detecting comprises performing microarray analysis, polymerase chain reaction (PCR), or sequencing.
  • PCR polymerase chain reaction
  • the PCR is a real-time quantitative PCR.
  • sample is blood, plasma, serum, saliva, urine, and/or cervical mucus.
  • sample volume is selected from the volume of less than 1ml, 750 pi, 500 m ⁇ , 250 m ⁇ , 200 m ⁇ , 175 m ⁇ , 150 m ⁇ , 125 m ⁇ , 100 m ⁇ , 75 m ⁇ , 50 m ⁇ , and 25 m ⁇ , and a range of 25-500 m ⁇ , 25-250 m ⁇ , 50-125 m ⁇ , and 100-250 m ⁇ .
  • the biological sample is processed within 1 hour, within 24 hours, or within 48 hours.
  • biological sample is incubated or mixed with a preservative.
  • the biological sample is stored at ambient temperature for at least 12, 18, 24, 36 or 48 hours, 12-48 hours, 18-48 hours, 18-36 hours, or 18-24 hours, before processing, and wherein the biological sample is whole blood.
  • the whole blood is capillary blood.
  • method comprises: a) obtaining or having obtained a blood sample from a pregnant subject, wherein the volume of blood is 80-10,000 pi, 80-5,000 m ⁇ , or 80-1,000 m ⁇ of venous blood, or 80-500 m ⁇ , or 80-250 m ⁇ of capillary blood; b) mixing the blood sample with a cell-free nucleic acid preservative; c) storing the blood sample for at least 12, 18, 24, 36 or 48 hours, 18-48 hours, 18-36 hours, or 18-24 hours, at ambient temperature; d) collecting 40-5,000 m ⁇ , 40-2,500 m ⁇ , or 40-500 m ⁇ of plasma from the venous blood sample, or 40-250 m ⁇ or 40-125 m ⁇ of plasma from the capillary blood sample; e) isolating cell- free nucleic acids from the plasma; f) detecting Y-chromosome DNA in the cell-free nucleic acids by performing quantitative PCR with all or a portion of the isolated cell-free nucleic
  • the method further comprises g) determining the sex of a fetus as male based on the detection of Y-chromosome DNA in the cell-free nucleic acids, or female based on the absence of detection of Y-chromosome DNA in the cell-free nucleic acids.
  • the volume of blood is 80-500 m ⁇ of capillary blood, wherein 40-250 m ⁇ of plasma is collected, wherein the gestational age of the fetus is 42-53 days and the accuracy of the test is at least 99%.
  • the sample is incubated or mixed with a cell-free nucleic acid preservative, wherein the preservative is an anti-coagulant (e.g., ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(P-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA), heparin), an antimicrobial, a sugar, and/or an amino acid.
  • the sample is a capillary blood sample collected from the upper arm of the subject.
  • the method comprises decontaminating the sample collection site.
  • the sample collection site is not a hand or finger.
  • the method does not comprises decontaminating the sample collection site.
  • the subject is human.
  • kits for collecting a biological sample from a pregnant subject for determining fetal sex comprising a blood collection tube, a lancet or a device for obtaining venous or capillary blood from the subject, a tourniquet, a bandage, an alcohol swab, optionally a nail or skin brush, and instructions.
  • the kit further comprises a decontaminating agent.
  • the decontaminating agent is bleach, an alcohol wipe, chlorhexidine gluconate, hydrogen peroxide, and/or iodine.
  • the instructions provide for sample collection at gestational age of 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks.
  • the subject is human
  • nucleic acid includes a plurality of such nucleic acids
  • composition is a reference to one or more compositions and to equivalents thereof known to those skilled in the art, and so forth.
  • the disclosure relates, in part, to the discovery that the sex of a fetus can be determined with high accuracy at very early gestational ages by detecting Y-chromosome DNA in a biological sample obtained from a pregnant subject, e.g., 4 weeks to 8 weeks gestational age.
  • the sex of a fetus can be determined with high accuracy, for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, at very early gestational ages, for example not more than 56, 53, 49, 46, 42, 39, 35, 32, or 28 days gestational age, or a range defined by any two of the preceding values, for example 35-53 days, 42-53 days, or 42-49 days.
  • the disclosure is based, in part, on the discovery of unexpected improvements in sensitivity, specificity, and accuracy of methods, compositions, and kits of the disclosure for determining the sex of a fetus in biological samples from a pregnant subject, e.g., greater than 99% accuracy at determining fetal sex in a blood sample obtained from a pregnant subject at 6 weeks gestation, or earlier.
  • the disclosure demonstrates that fetal nucleic acids present in the maternal circulation may be detected at very early time points in pregnancy to determine fetal sex in a subject.
  • the disclosure provides methods for determining fetal sex in a pregnant subject. In some embodiments, the methods determine fetal sex in the subject with at least 99% accuracy.
  • the methods determine fetal sex in the subject with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% accuracy, including 100% accuracy, or a range defined by any two of the preceding values, for example 98%-100%, 99.0%-100%.
  • compositions for use in the methods described herein may include one or more of compounds, primers, probes, preservatives, including anticoagulants, cell fixatives, protease inhibitors, phosphatase inhibitors, proteins, DNA or RNA preservatives.
  • kits for collecting biological samples from pregnant subjects or for determining fetal sex in a subject comprise one or more of a blood collection tube, a lancet or a device useful for obtaining venous or capillary blood from the subject, a tourniquet, a bandage, an alcohol swab, a nail or skin brush, and instructions for using the kits.
  • the disclosure provides methods, compositions, and kits for determining the sex of a fetus in a pregnant subject.
  • the methods of the disclosure involve the detection of Y- chromosome DNA in a biological sample obtained from a pregnant subject.
  • a biological sample comprising fetal nucleic acids may be obtained from a pregnant subject.
  • the biological sample obtained from the subject is typically blood, but can be any sample from bodily fluids, tissue or cells comprising the nucleic acids to be analyzed.
  • the biological sample may include, but is not limited to, whole blood, serum, plasma, urine, a cervical swab, saliva, a buccal swab, and/or amniotic fluid.
  • the biological sample of the disclosure can be obtained from blood.
  • about 0.1-10 mL of blood is obtained from a subject.
  • about 10-50 mL of blood is obtained from a subject.
  • the amount of blood obtained from the subject is, is about, is at least, is at least about, is not more than, or is not more than about, 40, 50, 60, 70, 80, 90, 100, 250, 500, 750, or 1000 pi, or 1, 5, or 10 ml, or a range defined by any two of the preceding values, for example, 1-500 m ⁇ , 50-1000 m ⁇ , 50 m ⁇ - 5 ml, and 100 m ⁇ - 10 ml.
  • Blood can be obtained from any suitable area of the body, including an arm, a leg, a finger, or blood accessible through a central venous catheter.
  • blood is collected from the finger using a lancet.
  • blood is collected from the arm via venipuncture.
  • blood is collected from the arm using a TAP device (Seventh Sense Biosystems, MA) or a Tasso-SST device (Tasso Inc., WA).
  • the site of blood collection is a site less likely to be contaminated with foreign DNA.
  • the site of blood collection is on the torso (e.g. stomach, side, back, shoulder), hip, upper leg (e.g.
  • the site of blood collection is the upper arm (i.e., located between the shoulder joint and elbow joint). In some embodiments, the site of blood collection is not the finger, or not the hand. In some embodiments the blood is not venous blood. In some embodiments, blood is collected following a treatment or activity. For example, blood can be collected following a medical exam. The timing of collection can also be coordinated to increase the amount of fetal nucleic acids present in the sample. For example, blood can be collected following exercise.
  • Blood may be combined with various components following collection to preserve or prepare samples for subsequent techniques.
  • blood is treated with an anticoagulant, a cell fixative, a protease inhibitor, a phosphatase inhibitor, a protein, a DNA, or an RNA preservative following collection.
  • the biological sample is incubated with a preservative.
  • the preservative is an anti-coagulant (e.g., ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'- tetraacetic acid (EGTA), heparin), an antimicrobial (e.g., imidazolidinyl urea), a sugar, and/or an amino acid.
  • the preservative is a solid, a liquid, and/or a gel.
  • blood is collected via venipuncture using vacuum collection tubes containing an anticoagulant such as EDTA, EGTA, or heparin. Blood can also be collected using a heparin-coated syringe and hypodermic needle. Blood can also be combined with components that will be useful for subsequent analysis of the fetal nucleic acids contained therein.
  • the volume of the biological sample obtained from the subject may be KM to 10ml.
  • the volume of the sample used to detect Y-chromosome DNA is a microvolume.
  • the microvolume is about l,000ul, about 900ul, about 800ul, about 700 ul, about 600ul, about 500ul, about 400ul, about 300ul, about 200ul, about 150ul, about lOOul, about 50ul, about 25ul, about lOul.
  • Blood samples are typically processed within a few hours from the time of collection to prevent significant degradation of the nucleic acids by enzymes present in blood. The methods of the disclosure enable the biological sample to be processed up to several months after being collected from the subject.
  • the biological sample is processed within 1 hour, within 24 hours, or within 48 hours. In other embodiments, the biological sample is not processed for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 1 week, at least, 2 weeks, or at least 4 weeks.
  • the biological sample should be free of contaminating DNA from a non-maternal or non-fetal source (e.g., touch DNA from another person).
  • a non-maternal or non-fetal source e.g., touch DNA from another person.
  • the presence or absence of Y-chromosome DNA in the biological sample is used to determine if a fetus is male or female.
  • Contaminating Y-chromosome DNA i.e., non-fetal Y-chromosome DNA
  • maternal blood is collected from a site on the body which is generally free of contaminating Y- chromosome DNA.
  • the site of blood collection is the upper arm.
  • the site of blood collection is on the torso (e.g. stomach, side, back, shoulder), hip, upper leg (e.g. thigh), upper arm, lower leg (e.g. calf). In some embodiments, the site of blood collection is not the finger, or not the hand.
  • a TAP blood specimen collection device is used to collect a maternal blood sample.
  • the TAP Blood Collection Device is a single-use, sterilized blood collection and transportation device that uses a combination of two mechanisms, capillary action and vacuum extraction.
  • the device consists of an integrated reservoir with a visual fill indicator window. The device is designed to collect and contain approximately 100-500 pL of capillary whole blood.
  • the internal fluid path is coated with lithium heparin, EDTA, EGTA, or other anticoagulants and/or preservatives.
  • the top of the device includes a green button or plunger and a fill indicator window.
  • the base of the device includes a release liner that covers a layer of hydrogel adhesive. The hydrogel adhesive seals to the skin and holds the device in place during use.
  • the TAP device contains an array of microneedles in order to puncture through the skin. The microneedles are activated by a spring, released by pushing a button or plunger on the device.
  • the device is provided sterile in a tray or foil pouch.
  • a preservative or cell stabilizer can optionally be used in the TAP device to allow for DNA analysis more than 6 hours after a blood sample is collected.
  • the blood sample is processed 6 hours, 8 hours, 10 hours, 12 hour, 24 hours, 48 hour, 72 hours, 96 hours, 120 hours, 144 hours, or 168 hours after collection.
  • the blood is stabilized in the TAP device with a preservative such that DNA concentration in the plasma portion of the blood sample remain relatively constant for up to 7 to 14 days post collection.
  • the preservative in the TAP device prevents significant genomic DNA contamination in blood samples for up to 7 to 14 days post collection.
  • the blood is stabilized in the TAP device with a preservative such that DNA concentration in the plasma portion of the blood sample remain relatively constant for up to 3 to 7 days post collection.
  • the site of blood collection is a site less likely to be contaminated with foreign DNA.
  • the site of blood collection is on the torso (e.g. stomach, side, back, shoulder), hip, upper leg (e.g. thigh), lower leg (e.g. calf).
  • the site of blood collection is the upper arm (i.e., located between the shoulder joint and elbow joint).
  • the site of blood collection is not the finger, or not the hand.
  • the blood is not venous blood.
  • the TAP device of Tasso-SST device including a preservative, facilitates storage of the blood sample collected in the tube at room temperature for at least, or about 14 days without cell lysis and without cell-free nucleic acid degradation of the blood sample due to DNase and RNase activity after blood draw.
  • the disclosure provides methods, compositions, and kits for the early determination of the sex of a fetus in a pregnant subject.
  • the subject is a human subject.
  • the pregnancy may be the result of natural conception (i.e., a natural pregnancy) of result from use of assisted reproductive technology (e.g., in-vitro fertilization).
  • assisted reproductive technology e.g., in-vitro fertilization
  • the pregnant subject has used assisted reproductive technology (ART) to become pregnant.
  • the assisted reproductive technology is in-vitro fertilization, use of fertility medication (e.g., clomifene), ovulation induction, cryopreservation, and/or intracytoplasmic sperm injection.
  • the pregnant subject has a high-risk pregnancy.
  • the pregnant subject is a carrier of a sex-linked recessive disease or disorder.
  • the disclosure provides methods, compositions, and kits useful for determining fetal sex at various timepoints in pregnancy.
  • Gestational age is a measure of the age of a pregnancy which is taken from the beginning of the woman’s last menstrual period (LMP), or the corresponding age of the gestation as estimated by a more accurate method if available.
  • Such methods include adding 14 days to a known duration since fertilization (as is possible in in vitro fertilization), or by obstetric ultrasonography.
  • the gestational age of the fetus is 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, lOweeks, 11 weeks, or 12 weeks.
  • the gestational age of the fetus is 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days 42 days, 49 days, 56 days, 63 days, 70 days, 77 days, or 84 days, or a range defined by any of the preceding values, for example 35-53 days, or 42-49 days.
  • the methods, compositions, and kits of the disclosure may be used in assays to determine fetal sex in a pregnant subject. Fetal sex assay performance can be assessed by determining the assay’s sensitivity, specificity, area under the ROC curve (AUC), accuracy, positive predictive value (PPV), and negative predictive value (NPV). Disclosed herein are assays for determining fetal sex in a pregnant subject.
  • the performance of the assay may be based on sensitivity.
  • the sensitivity of an assay of the disclosure may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100%.
  • the performance of the assay may be based on specificity.
  • the specificity of an assay of the disclosure may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100%.
  • the performance of the assay may be based on area under the ROC curve (AUC).
  • AUC of an assay of the disclosure may be at least about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95.
  • the performance of the assay may be based on accuracy.
  • the accuracy of an assay of the present disclosure may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100%.
  • the performance of the methods, compositions, and kits of the disclosure have been determined in multiple populations. In some embodiments, the performance of the methods, compositions, and kits of the disclosure have been determined in a population of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, and/or 1,000 or more pregnant subjects. In certain aspects the accuracy of an assay of the disclosure is determined in a population of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, and/or 1,000 or more pregnant subjects.
  • the sensitivity of an assay of the disclosure is determined in a population of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, and/or 1,000 or more pregnant subjects.
  • the specificity of an assay of the disclosure is determined in a population of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, and/or 1,000 or more pregnant subjects.
  • the disclosure provides methods, compositions, and kits for detecting Y- chromosome nucleic acids in a biological sample obtained from a pregnant subject.
  • the Y-chromosome nucleic acids are cell-free fetal nucleic acids (e.g., cffDNA).
  • the Y-chromosome nucleic acids are genomic fetal nucleic acids from a fetal cell (e.g., gfDNA).
  • the disclosure also provides methods for isolating and concentrating fetal nucleic acids in biological samples from pregnant subjects.
  • the methods further comprise isolating fetal nucleic acids with a silica column or magnetic beads.
  • the methods further comprise concentrating the fetal nucleic acids in the biological sample using heat.
  • the methods further comprise isolating fetal nucleic acids by incubating the sample with an enzyme (e.g., proteinase K).
  • an enzyme e.g., proteinase K
  • the disclosure provides methods, compositions, and kits for detecting Y- chromosome nucleic acids in a biological sample from a pregnant subject.
  • the detecting comprises performing microarray analysis, polymerase chain reaction (PCR), or sequencing.
  • the PCR is quantitative PCR (qPCR) or digital PCR.
  • the qPCR is a TaqMan assay.
  • the PCR is a nested PCR, a duplex PCR, or a multiplex PCR.
  • the reaction volume of the PCR is 2ul, 3ul, 4ul, 5ul, lOul, 15ul, 20ul, 25ul, or 50ul.
  • the mastermix used in the PCR contains Uracil N Glycosylase (UNG).
  • compositions for detecting Y-chromosome nucleic acids in a biological sample are primers and/or probes that are capable of amplifying and detecting at least one target sequence on the Y-chromosome.
  • the probe set may comprise one or more polynucleotide probes.
  • Individual polynucleotide probes comprise a nucleotide sequence derived from the nucleotide sequence of the target sequences or complementary sequences thereof.
  • the nucleotide sequence of the polynucleotide probe is designed such that it corresponds to, or is complementary to the target sequences.
  • the polynucleotide probe can specifically hybridize under either stringent or lowered stringency hybridization conditions to a region of the target sequences.
  • the selection of the polynucleotide probe sequences and determination of their uniqueness may be carried out in silico using techniques known in the art, for example, based on a BLASTN search of the polynucleotide sequence in question against gene sequence databases, such as the Human Genome Sequence, UniGene, dbEST or the non-redundant database at NCBI.
  • the polynucleotide probe is complementary to a region of a target mRNA derived from a target sequence in the probe set.
  • Computer programs can also be employed to select probe sequences that may not cross hybridize or may not hybridize non- specific ally.
  • the polynucleotide target sequences of the disclosure may range in length from about 15 nucleotides to the full length of the target sequence on the Y-chromosome. In one embodiment of the disclosure, the polynucleotide target sequences are at least about 15 nucleotides in length. In another embodiment, the polynucleotide target sequences are at least about 20 nucleotides in length. In a further embodiment, the polynucleotide target sequences are at least about 25 nucleotides in length. In another embodiment, the polynucleotide target sequences are between about 15 nucleotides and about 500 nucleotides in length.
  • the polynucleotide target sequences are between about 15 nucleotides and about 450 nucleotides, about 15 nucleotides and about 400 nucleotides, about 15 nucleotides and about 350 nucleotides, about 15 nucleotides and about 300 nucleotides, about 15 nucleotides and about 250 nucleotides, about 15 nucleotides and about 200 nucleotides in length.
  • the probes are at least 15 nucleotides in length.
  • the target sequences are at least 15 nucleotides in length.
  • the target sequences are at least 20 nucleotides, at least 25 nucleotides, at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, at least 125 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 225 nucleotides, at least 250 nucleotides, at least 275 nucleotides, at least 300 nucleotides, at least 325 nucleotides, at least 350 nucleotides, at least 375 nucleotides in length.
  • the present disclosure further provides primers and primer pairs capable of amplifying target sequences on the Y-chromosome.
  • the nucleotide sequences of the primer set may be provided in computer-readable media for in silico applications and as a basis for the design of appropriate primers for amplification of one or more target sequences of the primer set.
  • Primers based on the nucleotide sequences of target sequences can be designed for use in amplification of the target sequences.
  • a pair of primers can be used.
  • the pairs of primers are usually chosen so as to generate an amplification product of at least about 25 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 300 nucleotides.
  • These primers may be used in standard quantitative or qualitative PCR-based assays to detect the presence of fetal Y-chromosome nucleic acids in the biological sample.
  • these primers may be used in combination with probes, such as TaqMan probes or molecular beacons in amplifications using real-time PCR.
  • the primers or primer pairs when used in an amplification reaction, specifically amplify at least a portion of a nucleic acid sequence of a target selected from the Y-chromosome.
  • the target sequences are present on the Y-chromosome in multiple locations. In certain embodiments, the target sequences are present in 2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, about 20, about 25, about 50, about 75, about 100, about 150, about 200, about 300, about 400, about 500, about 750, about 1,000 locations on the Y-chromosome.
  • the sensitivity of assays of the disclosure can be increased by detecting and/or amplifying target sequences that are present on the Y-chromosome in multiple locations.
  • Multiple primer pairs can be used in the methods of the disclosure.
  • a duplex or multiplex qPCR assay may be used to increase the detection limit of an assay of the disclosure.
  • a label can be attached to or incorporated into a probe or primer polynucleotide to allow detection and/or quantitation of a target polynucleotide representing the target sequence of interest.
  • the analysis of a plurality of target sequences on the Y-chromosome may be carried out separately or simultaneously with one test sample.
  • the target on the Y-chromosome is SRY, DYS, and/or DAZ.
  • An assay consisting of a combination of the target sequences referenced in the instant disclosure may be constructed. Such a panel may be constructed using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more or target sequences.
  • the analysis of a single target sequence or subsets of target sequences comprising a larger panel of Y-chromosome targets could be carried out with the methods described within the instant disclosure to optimize assay sensitivity or specificity in various settings.
  • the ratio of a target sequence on the Y-chromosome and a control sequence from an autosomal chromosome may be used in an algorithm of the disclosure for determining fetal sex.
  • the methods, compositions, and kits of the disclosure can be used to detect very small amounts of Y-chromosome DNA in a biological sample from a pregnant subject.
  • the methods of the disclosure can detect about 1 to 0.1 genomic equivalent of cffDNA in a sample, about 0.9 genomic equivalent of cffDNA in a sample, about 0.8 genomic equivalent of cffDNA in a sample, about 0.7 genomic equivalent of cffDNA in a sample, about 0.6 genomic equivalent of cffDNA in a sample, about 0.5 genomic equivalent of cffDNA in a sample, about 0.4 genomic equivalent of cffDNA in a sample, about 0.3 genomic equivalent of cffDNA in a sample, about 0.2 genomic equivalent of cffDNA in a sample, about 0.1 genomic equivalent of cffDNA in a sample.
  • the methods of the disclosure can detect a single copy of Y-chromosome DNA.
  • the detecting comprises performing microarray analysis, polymerase chain reaction (PCR), or sequencing.
  • the PCR is quantitative PCR (qPCR) or digital PCR.
  • the qPCR is a TaqMan assay.
  • the PCR is a nested PCR, a duplex PCR, or a multiplex PCR.
  • the reaction volume of the PCR is 2ul, 3ul, 4ul, 5ul, KM, 15ul, 20ul, 25ul, or 50ul.
  • the mastermix used in the PCR contains Uracil N Glycosylase (UNG).
  • the methods further comprise interpreting data generated when detecting the Y-chromosome DNA. In some embodiments, the interpreting is performed using a machine learning algorithm, a cycle-threshold (CT) algorithm, or artificial intelligence.
  • CT cycle-threshold
  • kits for collecting biological samples from pregnant subjects or for detecting Y-chromosome nucleic acids in a biological sample from a pregnant subject A variety of kits having different components are contemplated by the disclosure. Generally speaking, the kit will include the means for detecting Y-chromosome in a biological sample from a pregnant subject. In another embodiment, the kit will include means for collecting a biological sample and instructions for use of the kit contents. In certain embodiments, the kit comprises a means for enriching or isolating fetal nucleic acids in a biological sample. In further aspects, the means for enriching or isolating fetal nucleic acids comprises reagents necessary to enrich or isolate fetal nucleic acids from a biological sample.
  • kits of the disclosure may include instructions for decontaminating the site on the pregnant subject where the sample will be collected.
  • the decontamination is performed by applying bleach to the site of collection, by applying an alcohol wipe to the site of collection, by treating the site of collection with ultra-violet light, by applying chlorhexidine gluconate, hydrogen peroxide, and/or iodine to the site of collection, by applying a brush (e.g., a nail brush) to the site of the collection.
  • a brush e.g., a nail brush
  • kits for obtaining a biological sample from a pregnant subject may comprise a blood collection tube, a lancet or a device useful for obtaining venous or capillary blood from the subject, a tourniquet, a bandage, an alcohol swab, a nail or skin brush, and instructions for using the kits.
  • the kits further comprise a decontaminating agent.
  • the decontaminating agent is bleach, an alcohol wipe, chlorhexidine gluconate, hydrogen peroxide, and/or iodine.
  • the device for obtaining venous or capillary blood is a lancet (e.g., BD Microtainer contact-activated lancet), a syringe, and/or a push-button blood collection device (e.g., a TAP device).
  • the biological sample is collected into a tube, onto a card, and/or a swab.
  • Methods and kits of the disclosure can include instructions that provide a minimum gestational age or gestational age range for sample collection.
  • the minimum gestational age is 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, lOweeks, 11 weeks, or 12 weeks.
  • the gestational age is 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days 42 days, 49 days, 56 days, 63 days, 70 days, 77 days, or 84 days.
  • Example 1 Determination of Fetal Sex at 28 Days Gestation
  • Whole blood samples are collected from pregnant woman of gestational age 28 days. The samples are used to validate a fetal sex test described herein using circulating cell free fetal DNA (ccffDNA) in maternal plasma.
  • ccffDNA circulating cell free fetal DNA
  • RT-qPCR Real-time quantitative polymerase chain reaction
  • Isolated cell-free DNA (5ul) is dispensed into 96-well plates and reacted with a custom master mix for a final PCR reaction volume of 25ul per well.
  • Male cell-free DNA is detected using a multi-copy target sequence on the Y-Chromosome.
  • An autosomal control gene is detected to confirm that a sufficient amount of total cfDNA (maternal and fetal) was isolated from the sample.
  • the cycling conditions for the PCR are set at 10 min at 95°C to allow for the initial denaturation of DNA and polymerase activation, followed by 45 cycles of one minute at 60°C and 15 seconds at 95°C.
  • results show that a fetal sex test of the present disclosure can accurately determine fetal sex as early as 4 weeks into pregnancy (i.e., 4 weeks gestational age) with up to 100% accuracy. These results also show that the methods and compositions of the present disclosure are useful for determining fetal sex in early pregnancy (as early as 4 weeks gestational age). The results demonstrate that the disclosure provides methods, assays, kits, and compositions for detecting fetal nucleic acids and determining fetal sex in early pregnancy (e.g., 4 weeks gestation).
  • the purpose of this study was to evaluate the clinical performance of an assay of the disclosure for noninvasive prenatal testing (NIPT) to determine fetal sex at 8 weeks gestation.
  • NIPT noninvasive prenatal testing
  • One hundred and eight participants were enlisted for this study.
  • Fetal gestational age of participants ranged from 7.00-10.57 weeks at the time of sample collection.
  • Fetal sex test results were confirmed through subsequent sonographic evaluation after participants had reached at least 14 weeks gestation.
  • Isolated cell-free DNA (5ul) was dispensed into 96-well plates and reacted with a custom master mix for a final PCR reaction volume of 25ul per well.
  • Male cell-free DNA was detected using a multi-copy target sequence on the Y-Chromosome.
  • An autosomal control gene was detected to confirm that a sufficient amount of total cfDNA (maternal and fetal) was isolated from the sample.
  • the cycling conditions for the PCR were set at 10 min at 95°C to allow for the initial denaturation of DNA and polymerase activation, followed by 45cycles of one minute at 60°C and 15seconds at 95°C.
  • Fetal sex was determined for all 108 participants. In this study, there were 51 male bearing pregnancies and 57 female bearing pregnancies. The fetal sex test correctly identified all 51 male bearing pregnancies, demonstrating 100% sensitivity for Y-chromosome DNA detection. There were 0 false negatives resulting in a positive predictive value of 100%. The fetal sex test correctly identified 56 of 57 female bearing pregnancies. There was one false positive resulting in a negative predictive value of 98%. Four of the 108 samples initially yielded an inconclusive result. A second sample was collected from the four participants and a result was obtained on the second round of testing.
  • the average CT value for the Y-target sequence for plasma from male bearing pregnancies was 29.89 ranging from 28.09- 32.03. Total cfDNA for both male bearing pregnancies and female bearing pregnancies were comparable.
  • the average CT value for total DNA in male bearing pregnancies was 32.81, with a range of 31.13-34.63.
  • the average CT value for the female bearing pregnancies was 32.93, with a range of 29.97-35.15.
  • the fetal sex test was shown to be 99.1% accurate for fetal sex determination at 8 weeks gestation and 100% sensitive for male fetal cell-free DNA.
  • NIPT noninvasive prenatal testing
  • ART assisted reproductive technology achieved
  • Isolated cell-free DNA (5ul) was dispensed into 96-well plates and reacted with a custom master mix for a final qPCR reaction volume of 25ul per well.
  • Male cell-free DNA was detected using a multi-copy target sequence on the Y-Chromosome.
  • An autosomal control gene was detected to confirm that a sufficient amount of total cfDNA (maternal and fetal) was isolated from the sample.
  • the cycling conditions for the qPCR were set at 10 min at 95°C to allow for the initial denaturation of DNA and polymerase activation, followed by 45cycles of one minute at 60°C and 15 seconds at 95°C.
  • Fetal sex was determined for all 18 participants. In this study, there were 6 male bearing pregnancies and 12 female bearing pregnancies. The fetal sex test correctly identified all 6 male bearing pregnancies, demonstrating 100% sensitivity for Y-chromosome DNA detection. There were no false negatives resulting in a positive predictive value of 100%. The fetal sex test correctly identified 12 of 12 female bearing pregnancies. There were no false positives resulting in a negative predictive value of 100%.
  • the average CT value for the Y-target sequence for plasma from male bearing pregnancies was 30.55 ranging from 29.48-31.16. Total cfDNA for both male bearing pregnancies and female bearing pregnancies were comparable. The average CT value for total DNA in male bearing pregnancies was 31.75. The average CT value for the female bearing pregnancies was 31.82.
  • the fetal sex test was shown to be 100% accurate for fetal sex determination at 6 weeks gestation and 100% sensitive for male fetal cell-free DNA. These results were obtained using lOOul of maternal plasma. These results showed that the methods and compositions of the disclosure are useful for determining fetal sex as early as 6 weeks gestation using a microvolume amount of maternal plasma. These results further showed that the methods of the disclosure are useful for providing greater than 99% accurate fetal sex test results from biological samples stored at ambient temperature for 24-48 hours before processing.
  • Example 4 Determination of Fetal Sex at 8 Weeks Gestation Using a TAP Blood Collection Device
  • the purpose of this study was to evaluate the performance of a fetal sex assay at eight- and nine-weeks gestation in capillary whole blood samples collected with a TAP blood collection device. Twenty-six pregnant women were enrolled in this study. A capillary blood sample was collected from each participant at approximately 8 to 9 weeks gestation. Samples were processed and tested approximately 24-72 hours after collection. The accuracy of the test was assessed by comparing the qPCR test results from maternal blood samples to sonogram evaluations performed after 14 weeks gestation.
  • TAP blood collection device per the manufacturer’s instructions (Seventh Sense Biosystems, MA). TAP devices containing the blood samples were mailed to a clinical lab and the blood sample was removed from the device with a pipette and subsequently centrifuged at 1,600 g for 15 minutes to separate plasma from whole blood. Next, 50pL of the separated plasma was incubated with Proteinase K. Following proteinase K treatment, cfDNA was isolated from the plasma samples using a MagMAX Cell-Free DNA Isolation Kit (ThermoFisher) according to the manufacturer’s instructions.
  • MagMAX Cell-Free DNA Isolation Kit ThermoFisher
  • Real-time quantitative polymerase chain reaction was utilized to detect male cell-free fetal DNA as follows. Isolated cell-free DNA (5ul) was dispensed into 96-well plates and reacted with a custom master mix for a final qPCR reaction volume of 25ul per well. Male cell- free DNA was detected using a multi-copy target sequence on the Y-Chromosome. An autosomal control gene was detected to confirm that a sufficient amount of total cfDNA (maternal and fetal) was isolated from the sample. The cycling conditions for the qPCR were set at 10 min at 95°C to allow for the initial denaturation of DNA and polymerase activation, followed by 45cycles of one minute at 60°C and 15 seconds at 95°C.
  • CT cycle threshold
  • Fetal sex was determined for all 26 participants. The fetal sex test correctly identified the sex of the fetus in all 26 pregnancies, demonstrating 100% sensitivity and 100% specificity for Y-chromosome DNA detection and 100% accuracy for fetal sex determination. There were no false positive or false negatives.
  • the fetal sex test was shown to be 100% accurate for fetal sex determination as early as 8 weeks gestation using 50ul of plasma collected with a TAP blood collection device. These results showed that the methods and compositions of the disclosure are useful for determining fetal sex as early as 8 weeks gestation using a microvolume amount of maternal plasma with a TAP blood collection device. These results further showed that the methods of the disclosure are useful for providing greater than 99% accurate fetal sex test results from biological samples stored at ambient temperature for 24-72 hours before processing.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés, des compositions et des kits pour la détermination précoce du sexe d'un foetus. L'invention concerne également des procédés, des compositions et des kits pour détecter des acides nucléiques foetaux dans des échantillons biologiques (par exemple de l'ADN foetal acellulaire).
EP20867331.9A 2019-09-23 2020-09-23 Procédés, compositions et kits pour déterminer le sexe d'un foetus Pending EP4034673A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201962903945P 2019-09-23 2019-09-23
US201962936329P 2019-11-15 2019-11-15
PCT/US2020/052187 WO2021061751A1 (fr) 2019-09-23 2020-09-23 Procédés, compositions et kits pour déterminer le sexe d'un foetus

Publications (2)

Publication Number Publication Date
EP4034673A1 true EP4034673A1 (fr) 2022-08-03
EP4034673A4 EP4034673A4 (fr) 2024-03-06

Family

ID=75167105

Family Applications (1)

Application Number Title Priority Date Filing Date
EP20867331.9A Pending EP4034673A4 (fr) 2019-09-23 2020-09-23 Procédés, compositions et kits pour déterminer le sexe d'un foetus

Country Status (5)

Country Link
US (2) US20220251650A1 (fr)
EP (1) EP4034673A4 (fr)
AU (1) AU2020354484A1 (fr)
CA (1) CA3155419A1 (fr)
WO (1) WO2021061751A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3700423A4 (fr) 2017-10-27 2021-08-18 Juno Diagnostics, Inc. Dispositifs, systèmes et procédés pour biopsie liquide à volumes ultra-faibles
WO2023010059A1 (fr) * 2021-07-27 2023-02-02 Gateway Genomics, Llc Procédés, compositions et kits pour la conservation d'acides nucléiques

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10335078B2 (en) 2014-08-04 2019-07-02 General Electric Company Device for separation and collection of plasma
WO2016161083A1 (fr) * 2015-03-31 2016-10-06 Theranos, Inc. Procédés, dispositifs, systèmes et kits pour prélèvement de sang automatisée au bout du doigt
ES2818569T3 (es) * 2015-09-09 2021-04-13 Drawbridge Health Inc Métodos para la recopilación, estabilización y conservación de muestras
WO2017214338A1 (fr) 2016-06-07 2017-12-14 Drawbridge Health, Inc. Procédés et dispositifs pour des molécules fortes ou stabilisantes
CN115192014A (zh) 2016-08-24 2022-10-18 贝克顿·迪金森公司 用于血液的附着流动的装置
GB2576635B (en) 2017-01-10 2020-08-05 Drawbridge Health Inc Devices, systems, and methods for sample collection
JP2020534873A (ja) * 2017-09-26 2020-12-03 ジュノ ダイアグノスティックス，インク． バイオマーカー解析のためのデバイス、システム、および方法
EP3803414A1 (fr) * 2018-06-01 2021-04-14 Laboratory Corporation of America Holdings Procédés et systèmes pour le génotypage protéomique lc-ms/ms

Also Published As

Publication number Publication date
AU2020354484A1 (en) 2022-05-19
US20230257811A1 (en) 2023-08-17
WO2021061751A1 (fr) 2021-04-01
US20220251650A1 (en) 2022-08-11
US11932907B2 (en) 2024-03-19
EP4034673A4 (fr) 2024-03-06
CA3155419A1 (fr) 2021-04-01

Similar Documents

Publication Publication Date Title
US11932907B2 (en) Fetal sex determination using capillary blood from upper arm
Liu et al. Endometrial microbiota in infertile women with and without chronic endometritis as diagnosed using a quantitative and reference range-based method
CN106906306B (zh) 快速并灵敏基因型鉴定及核酸检测
Obata-Yasuoka et al. A multiplex polymerase chain reaction–based diagnostic method for bacterial vaginosis
CN110093413A (zh) 检测β地中海贫血的引物组和试剂盒
RU2625006C1 (ru) Способ таргетной амплификации геномов возбудителей инфекций органов репродукции человека с целью одновременной идентификации возбудителей с набором праймеров
Liou et al. Fetal cells in the maternal circulation during first trimester in pregnancies
EP3257948A1 (fr) Procédé pour relier des résultats d'analyses diagnostiques rapides au point d'intervention à des procédés en laboratoire
Clausen et al. Noninvasive antenatal screening for fetal RHD in RhD negative women to guide targeted anti-D prophylaxis
Aghanoori et al. Sex determination using free fetal DNA at early gestational ages: a comparison between a modified mini-STR genotyping method and real-time PCR
Dupont et al. Fortuitous diagnosis of Trichomoniasis by PCR using panfungal primers
KR102505618B1 (ko) 신장이식 후 항체 매개성 거부반응의 진단을 위한 소변 엑소좀 유래 miRNA 유전자 바이오마커 및 이의 용도
AU2021400518A1 (en) Methods, compositions, and devices for the rapid determination of fetal sex
EP3591396B1 (fr) Trousse de détection d'infections sexuellement transmissibles asymptomatiques dans un échantillon d'urine
US20100129801A1 (en) Non-invasive recovery of rna and analysis of gene expression in skin
RU2659197C2 (ru) Способ выделения ДНК клинических изолятов Mycobacterium tuberculosis из ткани легкого
Tóth et al. PCR detection of Toxoplasma gondii in human fetal tissues
Aal-Hamdan et al. Fetal gender determination through Y-STR analysis of maternal plasma during the third trimester of pregnancy
EP3645737A1 (fr) Signatures de micro-arn sans cellules de mort de cellules bêta d'îlots de langerhans
CN107419009A (zh) 一种检测胃肠道间质瘤相关基因突变的试剂盒及其应用
Sulaiman et al. Comparison of nested and ELISA based polymerase chain reaction assays for detecting Chlamydia trachomatis in pregnant women with preterm complications.
Abd El Razik et al. Experimental study for human skin identification using a specific gene marker at different storage durations
Dodevska et al. ANALYSIS WITH PCR METHOD IN STIP
Hussein et al. Detection molecular for microdeletions of the Y chromosome in IVF technique
Issa et al. Diagnosis of Trichomonas vaginalis infection by PCR

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20220331

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 40079564

Country of ref document: HK

A4 Supplementary search report drawn up and despatched

Effective date: 20240205

RIC1 Information provided on ipc code assigned before grant

Ipc: G01N 33/48 20060101ALI20240130BHEP

Ipc: G01N 33/49 20060101ALI20240130BHEP

Ipc: C12Q 1/68 20180101ALI20240130BHEP

Ipc: C12Q 1/6876 20180101ALI20240130BHEP

Ipc: C12Q 1/6879 20180101ALI20240130BHEP

Ipc: C12Q 1/6806 20180101AFI20240130BHEP